Expanding genome editing tools through exploration of new CRISPR-Cas proteins and DNA repair enzymes ABSTRACT The current toolkit for human genome editing could be dramatically expanded through exploration of new CRISPR-Cas proteins that have yet to be characterized, and through the development of enzymatic tools to enable controlled repair of DNA breaks. We aim to advance the science and technology of genome editing by discovering and harnessing new CRISPR-Cas and related systems through a combination of bioinformatics and biochemistry. First, we will expand the diversity of CRISPR-CasX and -CasY sequences and analyze associated proteins, cofactors and small RNAs that contribute to the function of CasX and CasY enzymes. We will define the organization and functional components of these genomic loci, and conduct experiments with reconstituted systems that will establish mechanisms of action and enable testing in mammalian cells. Second, we will determine the mechanism of CRISPR-CasZ proteins, a newly discovered superfamily of RNA-guided enzymes. These proteins are exceptionally small, diverse, and have currently undefined functionalities for manipulating genomic sequences. Third, we will use protein family analyses that leverage huge datasets from uncultivated microbes and transcriptomics to discover and evaluate new enzymes for DNA manipulation, including helicases, nucleases, polymerases and recombinases. New gene variants will be identified based on protein family affiliation, functional predictions and genomic neighborhoods. We will establish assays for determining molecular activities including DNA and RNA binding, unwinding and cleavage. Together, the results to be obtained from these aims will lead to new insights into the functions of known proteins within CRISPR-Cas systems, and will likely uncover new kinds of proteins, enzymes and transcripts that contribute to genome surveillance and manipulation across the microbial world. This will lead to a much larger toolbox for genome editing than is currently available, enabling researchers and clinicians to quickly optimize technologies according to their needs.